Parallel analysis of thousands of biomolecules, like nucleic acids and proteins, and their specific properties is a challenge in biochemistry, genetics, medical diagnostics and microbiology.
Microarrays have proven useful in providing targeted DNA sequence information of the presence and concentration of many thousands of specific genetic regions in a single test. They can be used e.g. to identify novel genes, binding sites of transcription factors, changes in gene copy numbers, complex mutations in disease-causing human genes as well as to study microbial diversity in the environment. Microarrays also serve as a demultiplexing tool to sort spatially the sequence-tagged products of parallel reactions performed in solution (Stoughton et al., 2005). Microarrays can be printed from a set of pre-synthesized single-stranded DNA probes (oligonucleotides) or constructed using on-plate synthesis of the designed probes ({hacek over (S)}á{hacek over (s)}ik et al., 2004). In the technology patented by Illumina (Chee et al., 2006, 2007) microarrays comprise of thousands of microwells, each containing a single bead carrying 105 homologous probes. Beads are originally scattered randomly across the microarray and then decoded by sequential probing system, so that in the end each chip has a unique address file which encodes the identity of each bead. All these microarrays are analyzed using CCD-cameras or laser technology to detect fluorescence or chemiluminescence signals. Regardless of the current microarray technology, the raw data always comes in the scanned image, thus needing expensive high-resolution detectors and specific software for image analysis.
DNA sequencing technology has allowed analysis of millions of sequences in parallel (massively-parallel sequencing, also known as high-throughput sequencing or next-generation sequencing NGS) (e.g. Margulies et al., 2005, Rothberg & Leamon, 2008, Rothberg et al., 2011). Development of the new sequencing technology has largely replaced the need of prearranged microarrays. Still neither of the methods, high-throughput sequencing and microarray hybridization analysis, can alone offer a direct solution for the simultaneous analysis of the identity and labelling of a set of unknown nucleic acids. To study DNA-protein interactions, like histone modifications or transcription factor binding sites, chromatin immunoprecipitation (ChIP) can be used to separate targets of interest, based on the antibody precipitation and analysis of this way separated DNA using sequencing (ChIP-seq) or microarray technology (ChIP-chip) (reviewed by Hoffman & Jones, 2009). Another method for separating the functionally interesting nucleic acid from the sample is DNA stable isotope probing (DNA-SIP) by isolating heavier 13C-labelled DNA from the lighter unlabelled 12C-DNA by density-gradient ultracentrifugation (Radajewski et al., 2000, 2003). A recent microarray technique named as CHIP-SIP (Pett-Ridge et al., 2010, 2011; Mayali et al., 2011) has been introduced to analyse stable isotope labelled RNA hybridized on the pre-designed oligonucleotides on the microarray, with the detection using Nano-SIMS (nanoscale resolution secondary ionization mass spectrometer). Spatial distribution of radioactive objectives (like labelled biomolecules or microbes in tissue samples) can be determined by linking the microautoradiography image with in situ hybridization image in the protocol of MAR-FISH, microautoradiography-fluorescence in situ hybridization (Lee et al., 1999), which is done using fluorescence microscope imaging. “Isotope arrays” have been designed to study certain groups of substrate-consuming microorganisms by selecting their nucleic acids in a phylogenetic microarray by hybridization to phylogenetically designed oligonucleotides and determining the radioactivity of these microarray spots using autoradiographic x-ray film (Adamczyk et al., 2003, Wagner et al., 2006). In target enrichment platforms (like Agilent Sure select platform) nucleic acids with certain sequence motifs are separated by hybridization, then followed by sequencing of the separated nucleic acids. Target enrichment is useful for focusing the NGS workflow on key genomic regions of interest, while reducing cost per sample. Selecting target regions of interest e.g. only exon regions or expressed kinases enable thus more powerful and cost-efficient studies of genetic diversity.
Currently there are several manufacturers offering instruments for high-throughput sequencing (e.g. trademarks Illumina, 454/Roche, PacBio and Ion Torrent) of which Ion Torrent utilizes semiconductor technology and uses ion-sensitive, field-effect transistor (ISFET) arrays (Bergveld, 2003). Use of this technology, compatible with complementary metal-oxide semiconductor (CMOS) processes (Yeow et al., 1997) have enabled construction of miniaturized circuits in tiny microchips and constructing integrated circuits for low-cost sequencing devices (Rothberg et al., 2011). Sequencing using ISFETs is based on the measurement of the hydrogen ion concentration of a solution (commonly denoted as “pH”) (Rothberg & Hinz, 2009). In this technology sequence data are obtained by directly sensing the ions (pH) produced by template-directed DNA polymerase synthesis without light signal and image analysis. However, the semiconductor microchip has not been used for downstream analyses, like the analysis of radioactivity or chemiluminescence, as there has not been methods for the analysis of those parameters through the pH change.
Since the 18th century it has been known in photography that silver halides (like AgBr, AgI and AgCl) are light-sensitive forming metallic silver when exposed to light (Rieke, 2003). The silver-halide in light-sensitive photographic material is in small grains of size ˜1 μm, and photons of high enough energy can create a free photoelectrons, which can then reduce the silver ions into small atomic silver clusters, forming a “latent” image in these grains.
When the photographic material is then developed by a strongly reducing chemical agent, the reduced (latent) metallic silver cluster (even 3-4 Ag atoms) can catalyze the reduction of the entire grain, leading to a large number of 1010 to 1011 reduced metallic Ag atoms in a grain, and a tremendous intensification of the latent image. The photographic contrast arises, because during film development the reducing agent, typically hydroquinone, selectivity reduces only those grains, which contain atomic silver clusters, if the development process is performed under correct conditions (time, temperature and developer concentration). The developing agent in the developer solution is ionized, and these ions supply electrons to the silver ions of the exposed silver halide grains, reducing them to solid silver. Hydroquinone can also be replaced by ascorbic acid, or vitamin C, which, however, suffers from poor stability. This is because oxidation by-products formed during the development are acidic, retarding the development in and adjacent to areas of high activity. This also explains why ascorbate developers have poor keeping properties, as oxidised ascorbate is both ineffective as a developing agent and lowers the pH of the solution, making the remaining developing agents less active. Practical methods to improve the stability of ascorbate developer have been sought.
In autoradiography, instead of light, radioactivity is used for the direct sensitization of the light- and high-energy particle-sensitive material. An autoradiograph is an image on an x-ray film or on a nuclear emulsion (a photographic emulsion optimized for particle detection), produced by the pattern of decay emissions (e.g. alpha- or beta particles or gamma rays) from a distribution of a radioactive substance. Alternatively, the autoradiograph is also available as a digital image (digital autoradiography), due to the development of scintillation gas detectors (Barthe et al., 1999) or rare earth phosphorimaging systems (photostimulable phosphor plate).
When the autoradiography of radioactive material happens using x-ray film or nuclear emulsion for radiolabelled proteins, nucleic acids or entire organisms, a latent image is first formed just like in the exposure by light. However, when β-emitters (e.g. 14C, 3H, 35S, 32P, 33P) are used, the original high-energy electron and the secondary electrons it produces directly cause the reduction of the Ag ions to metallic silver (Erskine, 1979).
Autoradiography provides an alternative to direct radiation detection by solid state detectors such as Si p-n junctions, Li-drifted Si detectors or scintillation detectors (Knoll, 2010). The advantages of autoradiography over solid state detectors are: simplicity, reliability, stability, low cost, large dynamic range, and especially the fact that there is no count rate limitation, contrary to all types of solid-state detectors. It cannot, however, measure the emitted energy spectrum or work in real-time but in the present invention neither of these is required.
The present invention is directed to a problem how to detect radioactivity of multiple biomolecules in an array format. The solution provided by the invention is a method wherein the ion-sensitive field effect transistor array is incubated in a microautoradiographic emulsion so that radioactivity forms a latent image, which exponentially accelerates silver halide reduction to metallic silver when the emulsion is developed. This reduction will cause a pH decrease in unbuffered developer solutions (i.e. a developer with low buffer capacity), which can be measured using pH sensors provided by said array, thus providing information of thousands of separate biomolecules at the same time.